- Email: [email protected]
Biology of gut anaerobic fungi
BioSystems, 23 (1989) 5;3-64
53
Elsevier Scientific Publishers Ireland Ltd.
Biology of gut anaerobic fungi* Tom Bauchop Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, N.S.W. ~351 [Australia) (Received February 23rd, 1989) (Revision received July 7th, 1989)
The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant and ruminant-like animals and in the hindgut of hindgut fermenters. Comparative s Ludies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H 2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.
Keywords: Fungi; Rumen; Plant fibre; Cellulase, Digestion.
1. Introduction Microbial fermentation of ingested plant materials in the rumen is central to the digestion and nutrition of ruminants. As a result of extensive studies over several decades the rumen microbiota had been shown to comprise a dense complex population of microbes consisting predominantly of anaerobic bacteria and protozoa. In recent years, however, large populations of fungi, possessing the exceptional property of being obligate anaerobes, have been found to constitute an integral part of the rumen microbiota. These fungi colonise and grow on plant fragments in the rumen and are able to degrade cellulose and other plant s~ructural polysaccharides, functions at the crux of herbivore fermentative digestion. This major oversight in failure to detect these fungi may be explained by the fact that *Presented at the 7th Meeting of the International Society for Evolutionary Protistology (July 19-24, 1987).
microorganisms associated with the rumen plant fragments ("solids") have been little studied. In addition, it was standard practice in studies of rumen microbiology to use strained rumen contents and to discard the tureen "solids". The discarded "solids" fraction is now known to contain the thalli of tureen anaerobic fungi. Although the zoospores formed in the life cycle of the anaerobic rumen fungi, and present in strained tureen fluid, were undoubtedly observed, they were mistakenly classed with the tureen protozoan flagellates. Above all, the fundamentally aerobic nature of virtually all fungi had precluded serious consideration being given by rumen microbiologists to a role for fungi in the anaerobic environment of the rumen. In the past, common saprophytic fungi had been isolated from rumen contents but they were not regarded as indigenous rumen microbes (Clarke, 1977). These fungi were considered to be merely transients, as they continually enter the tureen on feed, are present in low numbers, and are unable to grow
0303-2647/89/$03.50 © 1089 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
54
under the anaerobic conditions of the rumen environment. The recently discovered rumen anaerobic fungi belong in an entirely different category - unlike all other known fungi they are obligate anaerobes. High populations are present in the rumen of cattle and sheep, and similar anaerobic fungi have been found in the gut of other large herbivorous animals - in the foregut of ruminant-like animals, and in the hindgut of animals possessing a hindgut fermentation. The essential features of these unusual fungi were revealed by two independent lines of study, initiated to investigate different and unrelated aspects of rumen microbiology. In the first of these studies Orpin (1974) investigated the possible sequestration on the rumen wall (Warner, 1966) of the flagellate Callimastix {NeocaUimastix) frontalis ( =N. patriciarum Orpin and Munn, 1986). Attempts to isolate the flagellate using a medium for isolation of trichomonads resulted in isolation of an organism bearing "a strong morphological resemblance to that of certain species of aquatic phycomycete fungi" (Orpin, 1975). The critical anaerobic culture conditions used in this work were not in accord with the basic tenet that fungi require oxygen for growth. Furthermore, the "NeocaUimastix flagellate" possessed up to fourteen flagella, compared with the maximum of two flagella recorded for zoosporic fungi. Orpin (1976, 1977a) subsequently isolated two additional species of these anaerobic organisms, Sphaeromonas communis Liebetanz and Piromonas communis Liebetanz. This represented an important achievement, as we now know that these two organisms together with Neocallimastix frontalis (Heath et al., 1983} represent the three major forms of anaerobic fungi known at present. It is no discredit to Orpin that considerable confusion existed about the exact nature of the organisms in a book chapter published at this time on rumen protozoa it was stated that "Neocallimastix frontalis has recently been shown to have fungal characteristics" (Clarke, 1977). Although the life-history of these organ-
isms had been determined in vitro from growth studies using a medium containing glucose as substrate, understanding of their develgpment in the rumen ecosystem remained unclear. Because zoospores of the rumen chytrids had been found to be present in low numbers in strained rumen fluid, they were believed to be unimportant members of the rumen microbiota. Sporangia (without rhizoids) of these fungi had been found mainly free in strained rumen fluid, and it was believed that they could exist in that state in rumen contents. However, in view of the low count of sporangia (< 3.6 × 104/cm3) in strained rumen fluid, it was concluded that the overall effect of these fungi on rumen metabolism was not great (Orpin, 1977b}. Understanding of the relationships of these fungi to rumen digesta plant fragments was also obscure. Although invasion of plant materials suspended in nylon bags in the sheep rumen was reported, it was found that when zoospores did attach they showed a preference for inflorescence tissues of feedstuffs (Orpin, 1977a,c; Orpin and Bountiff, 1978). However, it was appreciated that inflorescence tissues constituted only a minor component of normal diets. These anomalies were resolved as a result of a second line of investigation begun in 1976, Following the discovery, by means of scanning electron microscopy (SEM), of large populations of bacteria colonising the epithelium of sheep (Bauchop et al., 1975), it was decided to investigate rumen fibrous plant fragments by similar means to see if new information could be obtained on cellulolytic bacteria and fibre degradation. Initially this work resulted in new discoveries on the extent of protozoal colonisation of plant fragments in the rumen (Bauchop and Clarke, 1976; Bauchop, 1979a). In early 1978 this work took a new turn. In experiments where pieces of lucerne stem were suspended in nylon bags in a sheep rumen, large numbers of spherical to ovoid bodies were found attached to the stem vascular cylinder after short exposure
55 periods. These bodies were identified as an early developmental stage of the anaerobic fungi. The rapidity and extent of colonisation suggested a central role for fibrous plant materials in the life cycle of these fungi in the rumen and prompLed a search for them on natural rumen di~esta of cattle and sheep (Bauchop, 1979b). Ely means of SEM, the anaerobic fungi were shown to exist in large populations colonising fibrous plant fragments in the rumen of cattle and sheep. Their close association with the more refractory plant fragments in the rumen suggested clearly a possible role in fibre digestion, perhaps as initial colonisers in lignocellulose digestion (Bauchop, 1979b). Confirmation of cellulose digestion was obtained with pure cultures of fungi (Bauchop, 1979c; Orpin and Letcher, 1979) and later they were shown to carry out an active fermentation of cellulose (Bauchop and Mountfort, 1981). It is interesting that continued culture of the fungi in vitro with glucose as substrate can lead to loss of the ability to utilise many polysaccharides (Orpin and Letcher, 1979). Studies on the glyc,asidase activities of one of these fungi, N. fro~atalis, have demonstrated the presence of the array of extracellular enzymes required to degrade cellulose and hemicellulose to simple sugars (Pearce and Bauchop, 1985). Fungi similar to the rumen organisms have also been shown 1;o be present colonising plant fragments in the foregut or hindgut of herbivorous animals from different mammalian families, from diverse geographic locations (Bauchop, 1981, 1983). Thus it appears that anaerobic fungii may be ubiquitous in at least the larger forms of herbivorous animals. Although as yet these non-ruminant organisms have been litl;le studied their general properties are similar to those of the rumen fungi with regard to substrates utilized and fermentation products formed (T. Bauchop, unpublished). The discovery of ilarge populations of cellulolytic anaerobic J!ungi in the gut has extended our knowledge of the range of cellu-
lose-fermenting microbes present there. Current evidence suggests that the anaerobic fungi are important members of the rumen ecosystem with their preferential colonisation and growth on fibrous plant fragments contributing to degradation of fibrous feedstuffs. 2. Life cycle and classification
The life cycle of anaerobic rumen fungi consists of a stage involving a non-motile, vegetative, reproductive thallus and a stage where a motile flagellated zoospore is produced (Fig. 1). The mature vegetative thallus consists of an epibiotic sporangium and a much-branched rhizoid that grows within the tissues of the invaded plant fragment. The sporangium at maturity is ovoid and commonly about 100 ~m long. Zoospores are formed within the sporangium and are released by dissolution of part of the sporangium wall. Each zoospore can attach to a suitable plant fragment and develop into a new vegetative thallus. The development of a maintenance medium for anaerobic fungi (Joblin, 1981) permitted storage of the organisms for prolonged periods at 37°C. Many fungi were successfully stored for periods of up to 18 months (T. Bauchop, unpublished) which suggests the presence of a resting stage which had not yet been identified.
¢
Fig. 1.
Lifecycle of a commonrumen anaerobic fungus,
NeocaUimas fix fron talis.
56
The development of these fungi was first determined during growth in culture medium with glucose as substrate (Orpin, 1975). However, the key to understanding the life cycle in the rumen was the appreciation of the central role of rumen digesta plant fragments as the substrata for the colonisation, growth and development of the fungi (Bauchop, 1979b). In experiments with plant fragments suspended in nylon bags in the rumens of sheep, zoospores (Fig. 2) were found to invade plant fragments within 15 rain, and by 2 h large numbers had colonised exposed vascular cylinders of stem fragments (Fig. 3). In similar experiments with wheat straw leaf as substrate, development of rhizoids was demonstrated in 3 h by means of trypan-blue stain. The thinness of the leaf tissue together with the absence of masking effects of chlorophyll facilitated observation of rhizoids within the tissues of these stained
ii!¸ilili!i
/ i!!ii!!jii!i!iii!!i!i!ii!j/'
i!ii!~i~i~ii~ii~il~i~ii~ii ~:~i~ii~iil ~
%
Fig. 2.
Light micrograph of zoospore of NeocaUimastix
frontalis. Bar marker = 10 pm.
Fig. 3. SEM of early developmental stage of a rumen anaerobic fungus attached to lucerne stem fragment suspended in a sheep rumen for 2 h. Bar marker = 10 ~m.
preparations. Subsequent growth of these fungi over a period up to 24 h resulted in development of large rhizoids (Fig. 4) with extensive invasion of leaf tissues. The sporangium was found to develop from the original attached zoospore but did not begin to enlarge until about 12 h after settlement, although by this time rhizoid development was extensive. By approximately 24 h after settlement the sporangium reached maximum size. Empty sporangia found attached to plant fragments after 2 4 - 3 0 h in the rumen indicated the time span of the life cycle under conditions of once-daily feeding. An important step in setting the classification of these fungi on a firm basis was the assignment of the organism, N. frontalis, to a new family, the Neocallimasticaceae, in the Spizellomycetales of the Chytridiomycetes (Heath et al., 1983). Although some doubts remain about the phylogenetic affinities of Neocallimastix owing to the many unusual
57
Fig. 4. Light micrograph of rumen anaerobic fungus grown on wheat straw leaf in culture medium. Incubated 24 h at 39°C. Trypan blue stain. Bar marker = 100 ~m.
properties of th:is organism (Heath and Bauchop, 1985}, it is clear that the rumen anaerobic fungi should be classed with the more primitive aquatic fungi possessing relatively simple life cycles. The existence of a number of different fungi in the guts of cattle, sheep and other animals was indicated by morphological differences of zoospores and thalli, as well as by differences in their growth characteristics in pure cultures. However, detailed descriptions and classification of only a few of these fungi have been completed. Nevertheless at present only three major forms have been identified and the Neocallimasticaceae now contains three genera united by similarity in zoospore ultrastructure but separated on the basis of number of flagella on the zoospore and the rhizoid type (Gold et al., 1988). 3. Distribution of gut anaerobic fungi Most work has been carried out on domestic ruminants (cattle and sheep) and on the
fungi isolated from these animals, which has demonstrated worldwide distribution. However, similar fungi have been found in gut contents of herbivorous animals from different mammalian families and from wide geographical locations (Bauchop, 1979c, 1980). Present results indicate that fungal colonisation in herbivores requires a high-fibre diet and a capacious organ of fermentative digestion. Rumen fungi similar to those of domestic ruminants were found colonising plant fragments from the rumen of the feral ruminants, red deer (Cervus elaphus), reindeer (Rangifer tarandus) and impala (Aepyceros melampus). A range of ruminant-like animals also possess gut anaerobic fungi. Four species of feral macropod marsupials, the kangaroo tribe, had similar fungi colonising plant fragments in their stomachs. These species included the grey kangaroo (Macropus giganticus), redneck wallaby {M. rufogriseus), wallaroo (M. robustus robustus), and swamp wallaby (Wallabia bicolor). A fungus isolated from a walla-
58 TABLE I Populations of anaerobic fungi in the rumen of sheep and cattle on different feedstuffs. Animal
Diet
Fungal population .
Sheep Cattle/sheep
Wheat straw Meadow hay
4 4
Sheep Sheep Cattle/Sheep
Chaffed lucerne hay Pelleted lucerne Perennial ryegrass (pasture)
4 2 2
Sheep Sheep Sheep
Lotus pedunculatus (pasture)
1 N.D2
Sheep
Barley grain (85%) diet Continuously grazed pasture Young lucerne, red clover or white clover (pasture)
N.D. N.D.
"Fungal populations scored on a scale of I to 4 (high). bN.D. = Not detected by light microscopy or culture methods.
stomach contents was a Piromyces (=Piromonas) sp.. Anaerobic fungi were also isolated from a camel (Camelus dromedarius) and a llama (Lama glama), and belonged to roo
the genus Neocallimastix (both animals) and Piromyces (camel). Although these animals have a ruminant-like digestive system, as the fungi were isolated from fresh faecal samples the site of origin cannot be stated definitely. However, an origin in the stomach would seem most likely, as anaerobic fungi can likewise be isolated from faeces of cattle and sheep but there is no evidence for an extensive fungal flora in the hindgut of these animals. In two examples of herbivores possessing major hindgut fermentations, horses and elephants, large numbers of anaerobic fungi were found colonising plant stem fragments in freshly-voided faecal samples. Fungi isolated included Piromyces sp. and Caecomyces (=Sphaeromonas) sp. from the horses, and Piromyces sp. from elephants. The absence of Neocallimastix in the hindgut fermenters may be noteworthy.
Anaerobic fungi were not detected in fresh faeces of rabbits, possum (Trichosurus vulpecula), or man. However, several animals possessing a capacious foregut fermentative digestion would appear worthy of examination (Bauchop, 1977).
4. Colonisation of plant fragments Large populations of anaerobic rumen fungi attached to plant fragments were demonstrated first in the rumen of sheep and cattle by means of SEM (Bauchop, 1979b). Following the discovery that fibrous plant fragments were the major colonisation sites it was shown that the rumen fungal populations could be observed readily by means of light microscopy using either stained or unstained preparations of plant fragments from the rumen (Bauchop, 1980). Examination of plant fragments by either SEM or light microscopy revealed that highest populations of fungi were present in animals receiving stalky highfibre feeds (Fig. 5). The results of studies on animals fed a variety of different feedstuffs (Table 1) demonstrated that high-fibre diets supported the greatest populations of anaerobic fungi. With once-daily feeding, the fungi appeared to require approximately 24 h to complete their life-cycle (Bauchop, 1979b) and thus require substrates of long turnovertime in the rumen. Conversely the absence of fungi from the rumen of sheep ingesting soft leafy plants was explained by the short turnover-time (5--8 h) of these feeds in the rumen, resulting in failure of the fungi to maintain themselves. The major route of invasion by fungi, with all of the plant fragments studied, was via areas of epidermal damage. The hollow stems of graminaceous plants were colonised predominantly on the interior surfaces. Colonisation via stomata was observed also, but appeared comparatively unimportant with stem fragments of both legumes and grasses. However, stomatal invasion was found more frequently with leaves of grasses and was found most extensively with wheat straw
59
Fig. 5. SEM of sporangia of rumen anaerobic fungi attached to lucerne stem fragment from natural digesta of a sheep, 24 h after feedingwith chaffedlucerne hay. Bar marker = 50 ~m.
leaves. However, as with stem fragments, damaged areas of leaves were most heavily colonised. Hyphal penetration through stomata has also been reported to occur frequently with leaves of Digitaria pentzii Stent (Akin et al., 1983). The anaerobic rumen fungi were found associated mainly with vascular tissues of stems and leaves, 1;he plant tissues retained for the longest periods in the rumen. Stems were colonised principally with fungi attached to the vascular cylinders, both in legumes and grasses. However, it was clear from light microscopy studies that thin-walled tissues were colonised also (Bauchop, 1979b). Following fungal invasion, rhizoids eventually extended from the invasion sites to the vascular tissues where penetration and anchorage to the refractory vascular materials ensured extended retention in the rumen. Leaves
were found to be colonised by fungi also but the relatively soft leaves of lucerne were invaded to only a limited extent, and fungi were found attached mainly to vascular tissues. Leaves of grasses were found to be more heavily colonised than lucerne leaves. More refractory leaf material, such as wheat straw leaf, when suspended in nylon bags in the rumens of cattle and sheep for periods up to 24 h, was heavily colonised by anaerobic fungi, with areas around the leaf ribs being densely populated by fungi (Bauchop, 1979b).
5. Physiology of anaerobic fungi Information on the nutritional requirements of anaerobic rUmen fungi, although limited, indicates that they are relatively simple. Most early studies were carried out with media, formulated on ecological principles,
60
which contained rumen fluid. Lowe et al. {1985) described growth of fungi in complex media which lacked rumen fluid. More recently the first chemically-defined medium was described for the growth of Neocallimastix patriciarum (Orpin and Greenwood, 1986). This medium contained haemin, thiamin, dbiotin, ammonium ions, and a reduced source of sulphur (Orpin and Greenwood, 1986). An even simpler chemically-defined medium has now been found to maintain sustained growth through several subcultures of a variety of anaerobic fungi. These organisms (L. Douglas and T. Bauchop, unpublished) included N. frontalis PN1, Neocallimastix spp., PN2 and A1, Piromyces A2, and Caecomyces sp. A3. This medium contained haemin, ammonium ions, a reduced sulphur source (methionine or cysteine), and the substrate was microcystalline cellulose (L. Douglas and T. Bauchop, unpublished). It is of interest that in an earlier investigation it was reported that a deficiency of dietary sulphur limited the growth of anaerobic fungi in the rumen {Akin et al. 1983). Anaerobic rumen fungi were isolated readily using anaerobic roll-tube techniques with addition of antibiotics to the culture media. Pure cultures of the fungi were found to colonise and grow readily on fragments of any of the common fibrous feedstuffs incorporated into anaerobic culture media. Information on the enzymic and fermentative activities of the fungi has come from both in vitro and in vivo studies. Digestion of plant cell walls was demonstrated most clearly with wheat straw leaf as substrate and the extensive degradation involved digestion of cell walls of epidermal long cells (Bauchop, 1979c,1981). By comparison, adjacent epidermal short cells resisted digestion, presumably because they are highly silicified. In experiments with sheep fed the forage D. pentzii~ nylon bag digestibility studies showed that by 6 h fungi preferentially colonised the lignified cells of leaf blade sclerenchyma and by 24 h caused extensive degradation (Akin et al., 1983). Direct evidence for production of cellulase
was provided by culturing fungi on strips of filter paper {Bauchop, 1979c,1981; Orpin and Letcher, 1979). Initially, following zoospore attachment, the fungi developed as distinct colonies and the paper was digested only in areas of fungal growth. Successive sporulations resulted in extension of the area of colonisation and eventually, after approximately 5 days, the paper strips were almost completely digested with the paper fibres replaced by a semi-transparent mat of thalli. In similar experiments carried out with the rumen fungus, N. frontalis, the products of cellulose fermentation were found to be acetate, lactate, ethanol, formate, CO2 and H e (Bauchop and Mountfort, 1981) (Table 2). Hydrogen formation has not been reported previously in fungi, a further indication of the unusual nature of these microorganisms. A similar range of products, together with minor amounts of succinate, were produced by several different fungi isolated from a variety of animal species (T. Bauchop and G. Naylor, unpublished). The discovery that the fungi form stable cocultures with rumen methanogenic bacterium with resultant conversion of the fungal products H 2 and CO 2 to methane (Bauchop and Mountfort, 1981; Mountfort et al., 1982} {Table 2) provides an example of one form of TABLE
II
Fermentation of cellulose by Neocallimastix frontalis in the absence and presence of rumen H2-formate-utilising methanogenic bacteria. Product
Acetate Lactate Ethanol Formate Carbon dioxide Hydrogen Methane
mol/100 mol of hexose units Fungus alone
Fungus plus rumen methanogenic bacteria
72.7 67.0 37.4 83.1 37.6 35.3 0.0
34.7 2.9 19.0 1.0 88.7 < 0.5 58.7
61
microbial interactio~a that is probably common in the gut ecosystem. In the fungus-methanogen coculture, ce][lulose degradation commenced earlier, the rate was faster and the quantity digested was greater than in the monoculture (82 versus 53O/o). Recently the yield of cellulase was shown to be increased in cocultures compared with the monoculture (Mountfort and Asher, 1985). Anaerobic fungi have also been cultured on crystalline cellulose (Sigma), cotton, Avicel, carboxymethyl cellulose, cellophane, xylans and starch as well as on a range of simple sugars (Orpin and I,etcher 1979; Joblin, 1981; Pearce and Bauchop, 1985). Recently the cellular distribution and nature of the glycosidases produced by N. fr~,ntalis have been investigated (Pearce and Bauchop, 1985). Cellulosegrown fungal cultures were fractionated into extracellular, membrane and intracellular fractions and assayed for glycosidase activity using as substrates, Avicel, carboxymethyl cellulose, xylan, starch, polygalucturonic acid, and p-nitrophenyl-derivatives of galactose, glucose and xylose. Enzymic activity was highest in the extracellular fraction although the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards al][ substrates. Polygalacturonic acid was the only substrate not hydrolysed by the active fractions, indicating that pectinase was absent. Inability to ferment pectin or polygalacturonic acid has been found consistently in several as yet unclassified anaerobic fungi (T. Bauchop, unpublished). Of the major plant fibre components in forage, pectin is the most rapidly fermented by the rumen microbiota. The lack of pectinase in anaerobic fungi may thus relate to their requirement for materials of long retention time in the rumen, :needed for their survival there. The results of the glycosidase studies demonstrated that the common rumen fungus, N. frontalis, produces the range of enzymes necessary to degrade cellulose and hemicellulose to simple sugars (Pearce and Bauchop, 1985). Likewise all anaerobic fungi isolated in pure
culture on fibrous substrates have been found to grow on cellulose and xylan as substrates. The lipid compositions of P. communis and N. frontalis have been determined and shown to be broadly similar (Kemp et al., 1984; Body and Bauchop, 1985). The fatty acid compositions differ markedly from those of other members of Chytridiomycetes by the absence of long-chain polyenoic fatty acids and by the presence of a large proportion of a series of cis-monoenoic fatty acids between 14:1 and 24: 1. The absence of polyunsaturated fatty acids is explained by the anaerobic life style of these organisms. However, the formation of the large proportions of monoenoic fatty acids under anaerobic conditions constitutes a major difference which cannot he explained by known pathways of fatty acid biosynthesis in fungi (Body and Bauchop, 1985). This result indicates that biosynthesis occurs by an anaerobic pathway. Such a pathway has been found previously only in bacteria. A NeocaUimastix sp. has been shown to possess high proteolytic activity and the properties of the protease have been investigated (Wallace and Joblin, 1985). Source of anaerobic fungi Early experiments (Bauchop, 1979b) demonstrated that anaerobic fungi could not be cultured from ruminant feedstuffs although the fungi were readily cultured from rumen contents. Furthermore, sterile (autoclaved) wheat straw leaves, suspended in nylon bags in the rumen of cattle and sheep, were rapidly colonised by anaerobic fungi. It was concluded from these results that the fungi were indigenous tureen microbes. The question of the source of inoculation of newborn animals is identical to the question posed in the case of the rumen ciliates, which has been explained on the basis of close maternal contact. The broader question of the existence of anaerobic fungi in other environments, o f considerable interest to mycology, has also been studied (T. Bauchop, unpublished). Samples from a wide variety of anaerobic environments were
62
inoculated into anaerobic media containing plant materials (e.g. wheat straw leaf) as substrate, together with a mixture of penicillin and streptomycin. Inocula included samples of garden soil, pond, lake and river sediments, estuarine sediments, and samples from a sewage treatment plant. Cultures were incubated at room temperature (approx. 20°C) and 25°C for periods of up to several weeks, and fungi were isolated from garden soils and from sediments of a number of freshwater sources. Six different organisms, selected for further study, all proved to be facultative anaerobes, although they were grown under strict anaerobic conditions with regular transfers, for approximately one year. Unlike the gut anaerobes, these organisms all possessed a true mycelium and under anaerobic conditions and no other stages in their life-histories were detected. Culture inoculation appeared to be by transfer of mycelium. However, although sporangia and spores were produced under aerobic conditions, none of the organisms could be identified. N e v e r t h e l e s s these fungi did appear to possess unusual properties and when grown on agar plates, they grew down into the agar, and not on the surface. Thus obligately anaerobic fungi have only been found in gut contents of large herbivores, and despite an extensive search obligately anaerobic fungi could not be detected in other common anaerobic environments. Conclusion
The obligately anaerobic fungi of the gut have been found to be widely distributed in large herbivorous animals possessing primary fermentation in either the foregut or the hindgut. However, at present the fungi of domestic ruminants have been studied in greatest detail. The rumen anaerobic fungi have been shown to be active cellulolytic organisms that specifically colonise and grow on fibrous plant fragments. These properties taken together with the apparent extent of the rumen populations on plant fragments in animals
receiving high-fibre diets indicates a significant role for the fungi in fibre digestion. In addition, fungi may bring special properties to the degradation of plant materials in the rumen. As a result of their mode of growth involving hyphal extension, once attached to plant fragments, fungi can exert a force enabling them to penetrate deeply into tissues inaccessible to rumen bacteria or protozoa. Although it is clear that the anaerobic fungi are well equipped enzymically to contribute to rumen fermentation, the major question of the extent of this contribution remains to be answered. Unlike the rumen bacteria or protozoa the magnitude of the fungal populations cannot be measured by enumeration of any of the stages of their life cycle. Instead, it would seem necessary to determine the quantity of fungal tissues in the rumen in order to assess their importance, and this requires the development of a reliable method for determination of the fungal mass. The demonstration that anaerobic ecosystems are included in the range of environments which can be colonised by fungi is of considerable interest to microbiologists. At present obligately anaerobic fungi have not been found in anaerobic environments other than the herbivore gut contents. The anaerobic fungi also attract attention as a new group of cellulase-producing organisms which could be of value in biotechnology. Their ability to form stable coculture with methanogenic bacteria may also have significance for cellulose-degrading systems. The high cellulolytic activities of the unimproved cultures and the potential for increasing activities presents a challenge likely to be taken up, which could be significant in ruminant digestion or in biotechnology. At present there is considerable interest in genetic engineering of rumen microbes with a view to increasing productivity or overcoming problems of ruminant poisoning by toxic plants. While the rumen bacteria are receiving the main attention, it is clear that the challenge of the anaerobic fungi will be accepted also.
63
The fungi also offer considerable interest from the viewpoint of comparative biochemistry, particularly with regard to their fermentative mechanisms and the pathway of hydrogen production. Up until now it has been their colonisation of plant fibre and contribution to ruminant digestion which has attracted major attention. However, the many unusual properties of these fungi will undoubtedly stimulate much wider interest in the future. References Akin, D.E., Gordon, G.L.R. and Hogan, J.P., 1983, Rumen bacterial and fungal degradation of Digitaria pentzii grown with and without sulfur. Appl. Environ. Microbiol. 46, 738-748. Bauchop, T., 1977, Foregut fermentation, in: Microbiol Ecology of the Gut, R.T.J. Clarke and T. Bauchop (eds.) (Academic Press, New York) pp. 223-250. Bauchop, T., 1979a, The rumen ciliate Epidinium in primary degradation of plant tissues. Appl. Environ. Microbiol. 37, 1217- 1223. Bauchop, T., 1979b, Rumen anaerobic fungi of cattle and sheep. Appl. Environ. Microbiol. 38, 148--158. Bauchop, T., 1979c, The rumen anaerobic fungi: colonisers of plant fibre. Ann. F~ech. Vet. 10, 246--248. Bauchop, T., 1980, Scanning electron microscopy in the study of microbial digestion of plant fragments in the gut, in: Contempor~try Microbial Ecology, D.C. Ellwood, J.N., Hedger, M.J., Latham, J.M. Lynch and J.H, Slater (eds.) (Academic Press, New York) pp. 305 - 326. Bauchop, T., 1981, The anaerobic fungi in rumen fibre digestion. Agric. Environ. 6, 339-348. Bauchop, T., 1983, The gut anaerobic fungi: colonisers of dietary fibre, in: Fibre in Human and Animal Nutri-. tion, G. Wallace and L. Bell (eds.) (Royal Society of New Zealand) pp. 14:3--148. Bauchop, T. and Clarke, R.T.J.1976, Attachment of the ciliate Epidinium Crawley to plant fragments in the sheep rumen. Appl. Environ. Microbiol. 32, 417--422. Bauchop, T. and Mountfort, D.O., 1981, Cellulase fermentation by a rumev anaerobic fungus in both the absence and presence of rumen methanogens. Appl. Environ. Microbiol. 4,2, 1103--1100. Bauchop, T., Clarke, R.T.J. and Newhook, J.C., 1975, Scanning electron microscope study of bacteria associated with the rumen epithelium of sheep. Appl. Microbiol. 30, 668--f175. Body, D.R. and Bauchop, T., 1985, Lipid composition of an obligately anaerobic: fungus Neocallimastix frontalls isolated from a bovine rumen. Can. J. Microbiol. 31, 463-- 466.
Clarke, R.T.J., 1977, Protozoa in the rumen ecosystem, in: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauehop (eds.) (Academic Press, New York) pp. 251-275. Gold, J.J., Heath. I.B. and Bauchop, T., 1988, Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equii gen. nov., assigned to the NeocaUimasticaceae. Biosystems, 21, 403-415. Heath, I,B. and Bauchop, T., 1985, Mitosis and the phylogeny of the genus Neocallimastix~ Can. J Bot 63, 1595 - - 1604. Heath, I.B., Bauchop, T. and Skipp, R.A., 1983, Assignment of the rumen anaerobe, Neocallimastix frontalis, to the Spizellomyeetales (Chytridiomycetes) on the basis of its polyflagellate zoospore ultrastructure. Can. J . Bot. 61, 295-307. Joblin, K.N., 1981, Isolation, enumeration and maintenance of rumen anaerobic fungi in roll tubes. Appl. Environ. Microbiol. 42, 1119--1122. Kemp, P., Lander, D.L, and Orpin, C.G., 1984, The lipids of the rumen fungus Piromonus communis. J. Gen. Microbiol. 130, 2 7 - 37. Lowe, S.E., Theodorou, M.K., Trinci, A.P.J. and Hespell, R.B., 1985, Growth of anaerobic rumen fungi on defined and semi-defined media lacking rumen fluid. J. Gen. Microbiol. 131, 2225--2229. Mountfort, D.O. and Asher, R.A., 1985, Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 49, 1314--1322. Mountfort, D.O., Asher, R.A. and Baucbop, T., 1982, Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. strain RA1 and Methanosarcina barker~ Appl. Environ. Microbiol. 44, 128-134. Orpin, C.G. 1974, The rumen flagellate Callimastix frontalis: does sequestrastion occur? J. Gem Microbiol. 84, 395-- 398. 0rpin, C. G. 1975, Studies on the rumen flagellate NeocaUimastix frontalis. J. Gem Microbiol. 91, 2 4 9 262. Orpin, C. G., 1976, Studies on the rumen flagellate Sphaeromonas communis. J. Gen. Microbiol. 94, 2 7 0 - 280. Orpin, C.G., 1977a, The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen. J. Gem Microbiol. 99, 107--117. Orpin, C.G., 1977b, The occurrence of chitin in the cell walls of the rumen organisms Neocallimastix frontalis. Piromonas communis and Sphaeromonas communis. J. Gen. Microbiol. 99, 215-218. Orpin, C.G., 1977c, Invasion of plant tissues in the rumen by the flagellate NeocaUimastix frontalis. J. Gem Microbiol 98, 423-430. Orpin, C.G. and Bountiff, L. 1978, Zoospore chemotoxis in the rumen phycomycete NeocaUimastix frontalis. J. Gem Microbiol. 104, 113--122. Orpin, C.G. and Greenwood, Y., 1986, Nutritional and germination requirements of the rumen chytridiomy-
64 cete Neocallimastix patriciarum. Trans. Br. Mycol. Soc. 86, 103--109. Orpin, C.G. and Letcher, J., 1979, Utilization of cellulose, starch, xylan, and other hemicelluloses for growth by the rumen phycomycete Neocallimastix frontalis. Curr. Microbiol. 3, 121-124. Pearce, P.D. and Bauchop, T., 1985, Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates. Appl. Environ. Microbiol. 49, 1265--1269.
Wallace, R.J. and Joblin, K.N., 1985, Proteolytic activity of a rumen anaerobic fungus. FEMS Microbiol. Lett. 29, 19--25. Warner, A.C.I., 1966, Diurnal changes in the concentrations of micro-organisms in the rumens of sheep fed limited diets once daily. J. Gen. MicrobioL 45, 213-235.
53
Elsevier Scientific Publishers Ireland Ltd.
Biology of gut anaerobic fungi* Tom Bauchop Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, N.S.W. ~351 [Australia) (Received February 23rd, 1989) (Revision received July 7th, 1989)
The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant and ruminant-like animals and in the hindgut of hindgut fermenters. Comparative s Ludies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H 2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.
Keywords: Fungi; Rumen; Plant fibre; Cellulase, Digestion.
1. Introduction Microbial fermentation of ingested plant materials in the rumen is central to the digestion and nutrition of ruminants. As a result of extensive studies over several decades the rumen microbiota had been shown to comprise a dense complex population of microbes consisting predominantly of anaerobic bacteria and protozoa. In recent years, however, large populations of fungi, possessing the exceptional property of being obligate anaerobes, have been found to constitute an integral part of the rumen microbiota. These fungi colonise and grow on plant fragments in the rumen and are able to degrade cellulose and other plant s~ructural polysaccharides, functions at the crux of herbivore fermentative digestion. This major oversight in failure to detect these fungi may be explained by the fact that *Presented at the 7th Meeting of the International Society for Evolutionary Protistology (July 19-24, 1987).
microorganisms associated with the rumen plant fragments ("solids") have been little studied. In addition, it was standard practice in studies of rumen microbiology to use strained rumen contents and to discard the tureen "solids". The discarded "solids" fraction is now known to contain the thalli of tureen anaerobic fungi. Although the zoospores formed in the life cycle of the anaerobic rumen fungi, and present in strained tureen fluid, were undoubtedly observed, they were mistakenly classed with the tureen protozoan flagellates. Above all, the fundamentally aerobic nature of virtually all fungi had precluded serious consideration being given by rumen microbiologists to a role for fungi in the anaerobic environment of the rumen. In the past, common saprophytic fungi had been isolated from rumen contents but they were not regarded as indigenous rumen microbes (Clarke, 1977). These fungi were considered to be merely transients, as they continually enter the tureen on feed, are present in low numbers, and are unable to grow
0303-2647/89/$03.50 © 1089 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
54
under the anaerobic conditions of the rumen environment. The recently discovered rumen anaerobic fungi belong in an entirely different category - unlike all other known fungi they are obligate anaerobes. High populations are present in the rumen of cattle and sheep, and similar anaerobic fungi have been found in the gut of other large herbivorous animals - in the foregut of ruminant-like animals, and in the hindgut of animals possessing a hindgut fermentation. The essential features of these unusual fungi were revealed by two independent lines of study, initiated to investigate different and unrelated aspects of rumen microbiology. In the first of these studies Orpin (1974) investigated the possible sequestration on the rumen wall (Warner, 1966) of the flagellate Callimastix {NeocaUimastix) frontalis ( =N. patriciarum Orpin and Munn, 1986). Attempts to isolate the flagellate using a medium for isolation of trichomonads resulted in isolation of an organism bearing "a strong morphological resemblance to that of certain species of aquatic phycomycete fungi" (Orpin, 1975). The critical anaerobic culture conditions used in this work were not in accord with the basic tenet that fungi require oxygen for growth. Furthermore, the "NeocaUimastix flagellate" possessed up to fourteen flagella, compared with the maximum of two flagella recorded for zoosporic fungi. Orpin (1976, 1977a) subsequently isolated two additional species of these anaerobic organisms, Sphaeromonas communis Liebetanz and Piromonas communis Liebetanz. This represented an important achievement, as we now know that these two organisms together with Neocallimastix frontalis (Heath et al., 1983} represent the three major forms of anaerobic fungi known at present. It is no discredit to Orpin that considerable confusion existed about the exact nature of the organisms in a book chapter published at this time on rumen protozoa it was stated that "Neocallimastix frontalis has recently been shown to have fungal characteristics" (Clarke, 1977). Although the life-history of these organ-
isms had been determined in vitro from growth studies using a medium containing glucose as substrate, understanding of their develgpment in the rumen ecosystem remained unclear. Because zoospores of the rumen chytrids had been found to be present in low numbers in strained rumen fluid, they were believed to be unimportant members of the rumen microbiota. Sporangia (without rhizoids) of these fungi had been found mainly free in strained rumen fluid, and it was believed that they could exist in that state in rumen contents. However, in view of the low count of sporangia (< 3.6 × 104/cm3) in strained rumen fluid, it was concluded that the overall effect of these fungi on rumen metabolism was not great (Orpin, 1977b}. Understanding of the relationships of these fungi to rumen digesta plant fragments was also obscure. Although invasion of plant materials suspended in nylon bags in the sheep rumen was reported, it was found that when zoospores did attach they showed a preference for inflorescence tissues of feedstuffs (Orpin, 1977a,c; Orpin and Bountiff, 1978). However, it was appreciated that inflorescence tissues constituted only a minor component of normal diets. These anomalies were resolved as a result of a second line of investigation begun in 1976, Following the discovery, by means of scanning electron microscopy (SEM), of large populations of bacteria colonising the epithelium of sheep (Bauchop et al., 1975), it was decided to investigate rumen fibrous plant fragments by similar means to see if new information could be obtained on cellulolytic bacteria and fibre degradation. Initially this work resulted in new discoveries on the extent of protozoal colonisation of plant fragments in the rumen (Bauchop and Clarke, 1976; Bauchop, 1979a). In early 1978 this work took a new turn. In experiments where pieces of lucerne stem were suspended in nylon bags in a sheep rumen, large numbers of spherical to ovoid bodies were found attached to the stem vascular cylinder after short exposure
55 periods. These bodies were identified as an early developmental stage of the anaerobic fungi. The rapidity and extent of colonisation suggested a central role for fibrous plant materials in the life cycle of these fungi in the rumen and prompLed a search for them on natural rumen di~esta of cattle and sheep (Bauchop, 1979b). Ely means of SEM, the anaerobic fungi were shown to exist in large populations colonising fibrous plant fragments in the rumen of cattle and sheep. Their close association with the more refractory plant fragments in the rumen suggested clearly a possible role in fibre digestion, perhaps as initial colonisers in lignocellulose digestion (Bauchop, 1979b). Confirmation of cellulose digestion was obtained with pure cultures of fungi (Bauchop, 1979c; Orpin and Letcher, 1979) and later they were shown to carry out an active fermentation of cellulose (Bauchop and Mountfort, 1981). It is interesting that continued culture of the fungi in vitro with glucose as substrate can lead to loss of the ability to utilise many polysaccharides (Orpin and Letcher, 1979). Studies on the glyc,asidase activities of one of these fungi, N. fro~atalis, have demonstrated the presence of the array of extracellular enzymes required to degrade cellulose and hemicellulose to simple sugars (Pearce and Bauchop, 1985). Fungi similar to the rumen organisms have also been shown 1;o be present colonising plant fragments in the foregut or hindgut of herbivorous animals from different mammalian families, from diverse geographic locations (Bauchop, 1981, 1983). Thus it appears that anaerobic fungii may be ubiquitous in at least the larger forms of herbivorous animals. Although as yet these non-ruminant organisms have been litl;le studied their general properties are similar to those of the rumen fungi with regard to substrates utilized and fermentation products formed (T. Bauchop, unpublished). The discovery of ilarge populations of cellulolytic anaerobic J!ungi in the gut has extended our knowledge of the range of cellu-
lose-fermenting microbes present there. Current evidence suggests that the anaerobic fungi are important members of the rumen ecosystem with their preferential colonisation and growth on fibrous plant fragments contributing to degradation of fibrous feedstuffs. 2. Life cycle and classification
The life cycle of anaerobic rumen fungi consists of a stage involving a non-motile, vegetative, reproductive thallus and a stage where a motile flagellated zoospore is produced (Fig. 1). The mature vegetative thallus consists of an epibiotic sporangium and a much-branched rhizoid that grows within the tissues of the invaded plant fragment. The sporangium at maturity is ovoid and commonly about 100 ~m long. Zoospores are formed within the sporangium and are released by dissolution of part of the sporangium wall. Each zoospore can attach to a suitable plant fragment and develop into a new vegetative thallus. The development of a maintenance medium for anaerobic fungi (Joblin, 1981) permitted storage of the organisms for prolonged periods at 37°C. Many fungi were successfully stored for periods of up to 18 months (T. Bauchop, unpublished) which suggests the presence of a resting stage which had not yet been identified.
¢
Fig. 1.
Lifecycle of a commonrumen anaerobic fungus,
NeocaUimas fix fron talis.
56
The development of these fungi was first determined during growth in culture medium with glucose as substrate (Orpin, 1975). However, the key to understanding the life cycle in the rumen was the appreciation of the central role of rumen digesta plant fragments as the substrata for the colonisation, growth and development of the fungi (Bauchop, 1979b). In experiments with plant fragments suspended in nylon bags in the rumens of sheep, zoospores (Fig. 2) were found to invade plant fragments within 15 rain, and by 2 h large numbers had colonised exposed vascular cylinders of stem fragments (Fig. 3). In similar experiments with wheat straw leaf as substrate, development of rhizoids was demonstrated in 3 h by means of trypan-blue stain. The thinness of the leaf tissue together with the absence of masking effects of chlorophyll facilitated observation of rhizoids within the tissues of these stained
ii!¸ilili!i
/ i!!ii!!jii!i!iii!!i!i!ii!j/'
i!ii!~i~i~ii~ii~il~i~ii~ii ~:~i~ii~iil ~
%
Fig. 2.
Light micrograph of zoospore of NeocaUimastix
frontalis. Bar marker = 10 pm.
Fig. 3. SEM of early developmental stage of a rumen anaerobic fungus attached to lucerne stem fragment suspended in a sheep rumen for 2 h. Bar marker = 10 ~m.
preparations. Subsequent growth of these fungi over a period up to 24 h resulted in development of large rhizoids (Fig. 4) with extensive invasion of leaf tissues. The sporangium was found to develop from the original attached zoospore but did not begin to enlarge until about 12 h after settlement, although by this time rhizoid development was extensive. By approximately 24 h after settlement the sporangium reached maximum size. Empty sporangia found attached to plant fragments after 2 4 - 3 0 h in the rumen indicated the time span of the life cycle under conditions of once-daily feeding. An important step in setting the classification of these fungi on a firm basis was the assignment of the organism, N. frontalis, to a new family, the Neocallimasticaceae, in the Spizellomycetales of the Chytridiomycetes (Heath et al., 1983). Although some doubts remain about the phylogenetic affinities of Neocallimastix owing to the many unusual
57
Fig. 4. Light micrograph of rumen anaerobic fungus grown on wheat straw leaf in culture medium. Incubated 24 h at 39°C. Trypan blue stain. Bar marker = 100 ~m.
properties of th:is organism (Heath and Bauchop, 1985}, it is clear that the rumen anaerobic fungi should be classed with the more primitive aquatic fungi possessing relatively simple life cycles. The existence of a number of different fungi in the guts of cattle, sheep and other animals was indicated by morphological differences of zoospores and thalli, as well as by differences in their growth characteristics in pure cultures. However, detailed descriptions and classification of only a few of these fungi have been completed. Nevertheless at present only three major forms have been identified and the Neocallimasticaceae now contains three genera united by similarity in zoospore ultrastructure but separated on the basis of number of flagella on the zoospore and the rhizoid type (Gold et al., 1988). 3. Distribution of gut anaerobic fungi Most work has been carried out on domestic ruminants (cattle and sheep) and on the
fungi isolated from these animals, which has demonstrated worldwide distribution. However, similar fungi have been found in gut contents of herbivorous animals from different mammalian families and from wide geographical locations (Bauchop, 1979c, 1980). Present results indicate that fungal colonisation in herbivores requires a high-fibre diet and a capacious organ of fermentative digestion. Rumen fungi similar to those of domestic ruminants were found colonising plant fragments from the rumen of the feral ruminants, red deer (Cervus elaphus), reindeer (Rangifer tarandus) and impala (Aepyceros melampus). A range of ruminant-like animals also possess gut anaerobic fungi. Four species of feral macropod marsupials, the kangaroo tribe, had similar fungi colonising plant fragments in their stomachs. These species included the grey kangaroo (Macropus giganticus), redneck wallaby {M. rufogriseus), wallaroo (M. robustus robustus), and swamp wallaby (Wallabia bicolor). A fungus isolated from a walla-
58 TABLE I Populations of anaerobic fungi in the rumen of sheep and cattle on different feedstuffs. Animal
Diet
Fungal population .
Sheep Cattle/sheep
Wheat straw Meadow hay
4 4
Sheep Sheep Cattle/Sheep
Chaffed lucerne hay Pelleted lucerne Perennial ryegrass (pasture)
4 2 2
Sheep Sheep Sheep
Lotus pedunculatus (pasture)
1 N.D2
Sheep
Barley grain (85%) diet Continuously grazed pasture Young lucerne, red clover or white clover (pasture)
N.D. N.D.
"Fungal populations scored on a scale of I to 4 (high). bN.D. = Not detected by light microscopy or culture methods.
stomach contents was a Piromyces (=Piromonas) sp.. Anaerobic fungi were also isolated from a camel (Camelus dromedarius) and a llama (Lama glama), and belonged to roo
the genus Neocallimastix (both animals) and Piromyces (camel). Although these animals have a ruminant-like digestive system, as the fungi were isolated from fresh faecal samples the site of origin cannot be stated definitely. However, an origin in the stomach would seem most likely, as anaerobic fungi can likewise be isolated from faeces of cattle and sheep but there is no evidence for an extensive fungal flora in the hindgut of these animals. In two examples of herbivores possessing major hindgut fermentations, horses and elephants, large numbers of anaerobic fungi were found colonising plant stem fragments in freshly-voided faecal samples. Fungi isolated included Piromyces sp. and Caecomyces (=Sphaeromonas) sp. from the horses, and Piromyces sp. from elephants. The absence of Neocallimastix in the hindgut fermenters may be noteworthy.
Anaerobic fungi were not detected in fresh faeces of rabbits, possum (Trichosurus vulpecula), or man. However, several animals possessing a capacious foregut fermentative digestion would appear worthy of examination (Bauchop, 1977).
4. Colonisation of plant fragments Large populations of anaerobic rumen fungi attached to plant fragments were demonstrated first in the rumen of sheep and cattle by means of SEM (Bauchop, 1979b). Following the discovery that fibrous plant fragments were the major colonisation sites it was shown that the rumen fungal populations could be observed readily by means of light microscopy using either stained or unstained preparations of plant fragments from the rumen (Bauchop, 1980). Examination of plant fragments by either SEM or light microscopy revealed that highest populations of fungi were present in animals receiving stalky highfibre feeds (Fig. 5). The results of studies on animals fed a variety of different feedstuffs (Table 1) demonstrated that high-fibre diets supported the greatest populations of anaerobic fungi. With once-daily feeding, the fungi appeared to require approximately 24 h to complete their life-cycle (Bauchop, 1979b) and thus require substrates of long turnovertime in the rumen. Conversely the absence of fungi from the rumen of sheep ingesting soft leafy plants was explained by the short turnover-time (5--8 h) of these feeds in the rumen, resulting in failure of the fungi to maintain themselves. The major route of invasion by fungi, with all of the plant fragments studied, was via areas of epidermal damage. The hollow stems of graminaceous plants were colonised predominantly on the interior surfaces. Colonisation via stomata was observed also, but appeared comparatively unimportant with stem fragments of both legumes and grasses. However, stomatal invasion was found more frequently with leaves of grasses and was found most extensively with wheat straw
59
Fig. 5. SEM of sporangia of rumen anaerobic fungi attached to lucerne stem fragment from natural digesta of a sheep, 24 h after feedingwith chaffedlucerne hay. Bar marker = 50 ~m.
leaves. However, as with stem fragments, damaged areas of leaves were most heavily colonised. Hyphal penetration through stomata has also been reported to occur frequently with leaves of Digitaria pentzii Stent (Akin et al., 1983). The anaerobic rumen fungi were found associated mainly with vascular tissues of stems and leaves, 1;he plant tissues retained for the longest periods in the rumen. Stems were colonised principally with fungi attached to the vascular cylinders, both in legumes and grasses. However, it was clear from light microscopy studies that thin-walled tissues were colonised also (Bauchop, 1979b). Following fungal invasion, rhizoids eventually extended from the invasion sites to the vascular tissues where penetration and anchorage to the refractory vascular materials ensured extended retention in the rumen. Leaves
were found to be colonised by fungi also but the relatively soft leaves of lucerne were invaded to only a limited extent, and fungi were found attached mainly to vascular tissues. Leaves of grasses were found to be more heavily colonised than lucerne leaves. More refractory leaf material, such as wheat straw leaf, when suspended in nylon bags in the rumens of cattle and sheep for periods up to 24 h, was heavily colonised by anaerobic fungi, with areas around the leaf ribs being densely populated by fungi (Bauchop, 1979b).
5. Physiology of anaerobic fungi Information on the nutritional requirements of anaerobic rUmen fungi, although limited, indicates that they are relatively simple. Most early studies were carried out with media, formulated on ecological principles,
60
which contained rumen fluid. Lowe et al. {1985) described growth of fungi in complex media which lacked rumen fluid. More recently the first chemically-defined medium was described for the growth of Neocallimastix patriciarum (Orpin and Greenwood, 1986). This medium contained haemin, thiamin, dbiotin, ammonium ions, and a reduced source of sulphur (Orpin and Greenwood, 1986). An even simpler chemically-defined medium has now been found to maintain sustained growth through several subcultures of a variety of anaerobic fungi. These organisms (L. Douglas and T. Bauchop, unpublished) included N. frontalis PN1, Neocallimastix spp., PN2 and A1, Piromyces A2, and Caecomyces sp. A3. This medium contained haemin, ammonium ions, a reduced sulphur source (methionine or cysteine), and the substrate was microcystalline cellulose (L. Douglas and T. Bauchop, unpublished). It is of interest that in an earlier investigation it was reported that a deficiency of dietary sulphur limited the growth of anaerobic fungi in the rumen {Akin et al. 1983). Anaerobic rumen fungi were isolated readily using anaerobic roll-tube techniques with addition of antibiotics to the culture media. Pure cultures of the fungi were found to colonise and grow readily on fragments of any of the common fibrous feedstuffs incorporated into anaerobic culture media. Information on the enzymic and fermentative activities of the fungi has come from both in vitro and in vivo studies. Digestion of plant cell walls was demonstrated most clearly with wheat straw leaf as substrate and the extensive degradation involved digestion of cell walls of epidermal long cells (Bauchop, 1979c,1981). By comparison, adjacent epidermal short cells resisted digestion, presumably because they are highly silicified. In experiments with sheep fed the forage D. pentzii~ nylon bag digestibility studies showed that by 6 h fungi preferentially colonised the lignified cells of leaf blade sclerenchyma and by 24 h caused extensive degradation (Akin et al., 1983). Direct evidence for production of cellulase
was provided by culturing fungi on strips of filter paper {Bauchop, 1979c,1981; Orpin and Letcher, 1979). Initially, following zoospore attachment, the fungi developed as distinct colonies and the paper was digested only in areas of fungal growth. Successive sporulations resulted in extension of the area of colonisation and eventually, after approximately 5 days, the paper strips were almost completely digested with the paper fibres replaced by a semi-transparent mat of thalli. In similar experiments carried out with the rumen fungus, N. frontalis, the products of cellulose fermentation were found to be acetate, lactate, ethanol, formate, CO2 and H e (Bauchop and Mountfort, 1981) (Table 2). Hydrogen formation has not been reported previously in fungi, a further indication of the unusual nature of these microorganisms. A similar range of products, together with minor amounts of succinate, were produced by several different fungi isolated from a variety of animal species (T. Bauchop and G. Naylor, unpublished). The discovery that the fungi form stable cocultures with rumen methanogenic bacterium with resultant conversion of the fungal products H 2 and CO 2 to methane (Bauchop and Mountfort, 1981; Mountfort et al., 1982} {Table 2) provides an example of one form of TABLE
II
Fermentation of cellulose by Neocallimastix frontalis in the absence and presence of rumen H2-formate-utilising methanogenic bacteria. Product
Acetate Lactate Ethanol Formate Carbon dioxide Hydrogen Methane
mol/100 mol of hexose units Fungus alone
Fungus plus rumen methanogenic bacteria
72.7 67.0 37.4 83.1 37.6 35.3 0.0
34.7 2.9 19.0 1.0 88.7 < 0.5 58.7
61
microbial interactio~a that is probably common in the gut ecosystem. In the fungus-methanogen coculture, ce][lulose degradation commenced earlier, the rate was faster and the quantity digested was greater than in the monoculture (82 versus 53O/o). Recently the yield of cellulase was shown to be increased in cocultures compared with the monoculture (Mountfort and Asher, 1985). Anaerobic fungi have also been cultured on crystalline cellulose (Sigma), cotton, Avicel, carboxymethyl cellulose, cellophane, xylans and starch as well as on a range of simple sugars (Orpin and I,etcher 1979; Joblin, 1981; Pearce and Bauchop, 1985). Recently the cellular distribution and nature of the glycosidases produced by N. fr~,ntalis have been investigated (Pearce and Bauchop, 1985). Cellulosegrown fungal cultures were fractionated into extracellular, membrane and intracellular fractions and assayed for glycosidase activity using as substrates, Avicel, carboxymethyl cellulose, xylan, starch, polygalucturonic acid, and p-nitrophenyl-derivatives of galactose, glucose and xylose. Enzymic activity was highest in the extracellular fraction although the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards al][ substrates. Polygalacturonic acid was the only substrate not hydrolysed by the active fractions, indicating that pectinase was absent. Inability to ferment pectin or polygalacturonic acid has been found consistently in several as yet unclassified anaerobic fungi (T. Bauchop, unpublished). Of the major plant fibre components in forage, pectin is the most rapidly fermented by the rumen microbiota. The lack of pectinase in anaerobic fungi may thus relate to their requirement for materials of long retention time in the rumen, :needed for their survival there. The results of the glycosidase studies demonstrated that the common rumen fungus, N. frontalis, produces the range of enzymes necessary to degrade cellulose and hemicellulose to simple sugars (Pearce and Bauchop, 1985). Likewise all anaerobic fungi isolated in pure
culture on fibrous substrates have been found to grow on cellulose and xylan as substrates. The lipid compositions of P. communis and N. frontalis have been determined and shown to be broadly similar (Kemp et al., 1984; Body and Bauchop, 1985). The fatty acid compositions differ markedly from those of other members of Chytridiomycetes by the absence of long-chain polyenoic fatty acids and by the presence of a large proportion of a series of cis-monoenoic fatty acids between 14:1 and 24: 1. The absence of polyunsaturated fatty acids is explained by the anaerobic life style of these organisms. However, the formation of the large proportions of monoenoic fatty acids under anaerobic conditions constitutes a major difference which cannot he explained by known pathways of fatty acid biosynthesis in fungi (Body and Bauchop, 1985). This result indicates that biosynthesis occurs by an anaerobic pathway. Such a pathway has been found previously only in bacteria. A NeocaUimastix sp. has been shown to possess high proteolytic activity and the properties of the protease have been investigated (Wallace and Joblin, 1985). Source of anaerobic fungi Early experiments (Bauchop, 1979b) demonstrated that anaerobic fungi could not be cultured from ruminant feedstuffs although the fungi were readily cultured from rumen contents. Furthermore, sterile (autoclaved) wheat straw leaves, suspended in nylon bags in the rumen of cattle and sheep, were rapidly colonised by anaerobic fungi. It was concluded from these results that the fungi were indigenous tureen microbes. The question of the source of inoculation of newborn animals is identical to the question posed in the case of the rumen ciliates, which has been explained on the basis of close maternal contact. The broader question of the existence of anaerobic fungi in other environments, o f considerable interest to mycology, has also been studied (T. Bauchop, unpublished). Samples from a wide variety of anaerobic environments were
62
inoculated into anaerobic media containing plant materials (e.g. wheat straw leaf) as substrate, together with a mixture of penicillin and streptomycin. Inocula included samples of garden soil, pond, lake and river sediments, estuarine sediments, and samples from a sewage treatment plant. Cultures were incubated at room temperature (approx. 20°C) and 25°C for periods of up to several weeks, and fungi were isolated from garden soils and from sediments of a number of freshwater sources. Six different organisms, selected for further study, all proved to be facultative anaerobes, although they were grown under strict anaerobic conditions with regular transfers, for approximately one year. Unlike the gut anaerobes, these organisms all possessed a true mycelium and under anaerobic conditions and no other stages in their life-histories were detected. Culture inoculation appeared to be by transfer of mycelium. However, although sporangia and spores were produced under aerobic conditions, none of the organisms could be identified. N e v e r t h e l e s s these fungi did appear to possess unusual properties and when grown on agar plates, they grew down into the agar, and not on the surface. Thus obligately anaerobic fungi have only been found in gut contents of large herbivores, and despite an extensive search obligately anaerobic fungi could not be detected in other common anaerobic environments. Conclusion
The obligately anaerobic fungi of the gut have been found to be widely distributed in large herbivorous animals possessing primary fermentation in either the foregut or the hindgut. However, at present the fungi of domestic ruminants have been studied in greatest detail. The rumen anaerobic fungi have been shown to be active cellulolytic organisms that specifically colonise and grow on fibrous plant fragments. These properties taken together with the apparent extent of the rumen populations on plant fragments in animals
receiving high-fibre diets indicates a significant role for the fungi in fibre digestion. In addition, fungi may bring special properties to the degradation of plant materials in the rumen. As a result of their mode of growth involving hyphal extension, once attached to plant fragments, fungi can exert a force enabling them to penetrate deeply into tissues inaccessible to rumen bacteria or protozoa. Although it is clear that the anaerobic fungi are well equipped enzymically to contribute to rumen fermentation, the major question of the extent of this contribution remains to be answered. Unlike the rumen bacteria or protozoa the magnitude of the fungal populations cannot be measured by enumeration of any of the stages of their life cycle. Instead, it would seem necessary to determine the quantity of fungal tissues in the rumen in order to assess their importance, and this requires the development of a reliable method for determination of the fungal mass. The demonstration that anaerobic ecosystems are included in the range of environments which can be colonised by fungi is of considerable interest to microbiologists. At present obligately anaerobic fungi have not been found in anaerobic environments other than the herbivore gut contents. The anaerobic fungi also attract attention as a new group of cellulase-producing organisms which could be of value in biotechnology. Their ability to form stable coculture with methanogenic bacteria may also have significance for cellulose-degrading systems. The high cellulolytic activities of the unimproved cultures and the potential for increasing activities presents a challenge likely to be taken up, which could be significant in ruminant digestion or in biotechnology. At present there is considerable interest in genetic engineering of rumen microbes with a view to increasing productivity or overcoming problems of ruminant poisoning by toxic plants. While the rumen bacteria are receiving the main attention, it is clear that the challenge of the anaerobic fungi will be accepted also.
63
The fungi also offer considerable interest from the viewpoint of comparative biochemistry, particularly with regard to their fermentative mechanisms and the pathway of hydrogen production. Up until now it has been their colonisation of plant fibre and contribution to ruminant digestion which has attracted major attention. However, the many unusual properties of these fungi will undoubtedly stimulate much wider interest in the future. References Akin, D.E., Gordon, G.L.R. and Hogan, J.P., 1983, Rumen bacterial and fungal degradation of Digitaria pentzii grown with and without sulfur. Appl. Environ. Microbiol. 46, 738-748. Bauchop, T., 1977, Foregut fermentation, in: Microbiol Ecology of the Gut, R.T.J. Clarke and T. Bauchop (eds.) (Academic Press, New York) pp. 223-250. Bauchop, T., 1979a, The rumen ciliate Epidinium in primary degradation of plant tissues. Appl. Environ. Microbiol. 37, 1217- 1223. Bauchop, T., 1979b, Rumen anaerobic fungi of cattle and sheep. Appl. Environ. Microbiol. 38, 148--158. Bauchop, T., 1979c, The rumen anaerobic fungi: colonisers of plant fibre. Ann. F~ech. Vet. 10, 246--248. Bauchop, T., 1980, Scanning electron microscopy in the study of microbial digestion of plant fragments in the gut, in: Contempor~try Microbial Ecology, D.C. Ellwood, J.N., Hedger, M.J., Latham, J.M. Lynch and J.H, Slater (eds.) (Academic Press, New York) pp. 305 - 326. Bauchop, T., 1981, The anaerobic fungi in rumen fibre digestion. Agric. Environ. 6, 339-348. Bauchop, T., 1983, The gut anaerobic fungi: colonisers of dietary fibre, in: Fibre in Human and Animal Nutri-. tion, G. Wallace and L. Bell (eds.) (Royal Society of New Zealand) pp. 14:3--148. Bauchop, T. and Clarke, R.T.J.1976, Attachment of the ciliate Epidinium Crawley to plant fragments in the sheep rumen. Appl. Environ. Microbiol. 32, 417--422. Bauchop, T. and Mountfort, D.O., 1981, Cellulase fermentation by a rumev anaerobic fungus in both the absence and presence of rumen methanogens. Appl. Environ. Microbiol. 4,2, 1103--1100. Bauchop, T., Clarke, R.T.J. and Newhook, J.C., 1975, Scanning electron microscope study of bacteria associated with the rumen epithelium of sheep. Appl. Microbiol. 30, 668--f175. Body, D.R. and Bauchop, T., 1985, Lipid composition of an obligately anaerobic: fungus Neocallimastix frontalls isolated from a bovine rumen. Can. J. Microbiol. 31, 463-- 466.
Clarke, R.T.J., 1977, Protozoa in the rumen ecosystem, in: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauehop (eds.) (Academic Press, New York) pp. 251-275. Gold, J.J., Heath. I.B. and Bauchop, T., 1988, Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equii gen. nov., assigned to the NeocaUimasticaceae. Biosystems, 21, 403-415. Heath, I,B. and Bauchop, T., 1985, Mitosis and the phylogeny of the genus Neocallimastix~ Can. J Bot 63, 1595 - - 1604. Heath, I.B., Bauchop, T. and Skipp, R.A., 1983, Assignment of the rumen anaerobe, Neocallimastix frontalis, to the Spizellomyeetales (Chytridiomycetes) on the basis of its polyflagellate zoospore ultrastructure. Can. J . Bot. 61, 295-307. Joblin, K.N., 1981, Isolation, enumeration and maintenance of rumen anaerobic fungi in roll tubes. Appl. Environ. Microbiol. 42, 1119--1122. Kemp, P., Lander, D.L, and Orpin, C.G., 1984, The lipids of the rumen fungus Piromonus communis. J. Gen. Microbiol. 130, 2 7 - 37. Lowe, S.E., Theodorou, M.K., Trinci, A.P.J. and Hespell, R.B., 1985, Growth of anaerobic rumen fungi on defined and semi-defined media lacking rumen fluid. J. Gen. Microbiol. 131, 2225--2229. Mountfort, D.O. and Asher, R.A., 1985, Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 49, 1314--1322. Mountfort, D.O., Asher, R.A. and Baucbop, T., 1982, Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. strain RA1 and Methanosarcina barker~ Appl. Environ. Microbiol. 44, 128-134. Orpin, C.G. 1974, The rumen flagellate Callimastix frontalis: does sequestrastion occur? J. Gem Microbiol. 84, 395-- 398. 0rpin, C. G. 1975, Studies on the rumen flagellate NeocaUimastix frontalis. J. Gem Microbiol. 91, 2 4 9 262. Orpin, C. G., 1976, Studies on the rumen flagellate Sphaeromonas communis. J. Gen. Microbiol. 94, 2 7 0 - 280. Orpin, C.G., 1977a, The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen. J. Gem Microbiol. 99, 107--117. Orpin, C.G., 1977b, The occurrence of chitin in the cell walls of the rumen organisms Neocallimastix frontalis. Piromonas communis and Sphaeromonas communis. J. Gen. Microbiol. 99, 215-218. Orpin, C.G., 1977c, Invasion of plant tissues in the rumen by the flagellate NeocaUimastix frontalis. J. Gem Microbiol 98, 423-430. Orpin, C.G. and Bountiff, L. 1978, Zoospore chemotoxis in the rumen phycomycete NeocaUimastix frontalis. J. Gem Microbiol. 104, 113--122. Orpin, C.G. and Greenwood, Y., 1986, Nutritional and germination requirements of the rumen chytridiomy-
64 cete Neocallimastix patriciarum. Trans. Br. Mycol. Soc. 86, 103--109. Orpin, C.G. and Letcher, J., 1979, Utilization of cellulose, starch, xylan, and other hemicelluloses for growth by the rumen phycomycete Neocallimastix frontalis. Curr. Microbiol. 3, 121-124. Pearce, P.D. and Bauchop, T., 1985, Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates. Appl. Environ. Microbiol. 49, 1265--1269.
Wallace, R.J. and Joblin, K.N., 1985, Proteolytic activity of a rumen anaerobic fungus. FEMS Microbiol. Lett. 29, 19--25. Warner, A.C.I., 1966, Diurnal changes in the concentrations of micro-organisms in the rumens of sheep fed limited diets once daily. J. Gen. MicrobioL 45, 213-235.